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Background: The ability of honeybees to survive and sustain honey production depends on their ability to adapt to their environment. This study evaluated the seasonal fluctuations of antioxidant activities and biochemical compositions of honeybees, Apis mellifera adansoniiL. from rainforest, guinea savannah and derived savannah zones of Nigeria during three seasonal periods (onset of rain, wet season and dry season) from January to December, 2017. Honeybees were collected from fully colonized colonies of two different apiaries from each of the ecological zones during the seasonal periods. Amino acid composition was determined using high-performance liquid chromatography. Antioxidant enzymes activity, body protein, glucose and lipid profile were evaluated using standard protocols. Results: Seasonal periods showed significant (p< 0.05) effect on the eighteen amino acids, superoxide dismutase (SOD), triglycerides and fat-free nitrogen recorded in the honeybees. Amino acids were higher in the honeybees from the rainforest zone, regardless of the seasonal period. SOD and body protein were higher during the wet season in the three ecological zones. However, seasonal periods showed no significant (p> 0.05) influence on the body glucose, protein pattern, total cholesterol, activities of malondialdehyde, reduced glutathione, glutathione peroxidase and catalase. Body glucose content in the honeybees from the rainforest and guinea savannah zones was highest during the dry season. Conclusions: Antioxidant activities and biochemical compositions of honeybees are differently affected by both ecological zones and seasonal fluctuations.